1. Field of Invention
This invention relates to an improved process for synthesis of peptides by the solid phase method. More particularly, the invention relates to the synthesis of peptide amides by solid phase peptide synthesis using a novel resin support, methylbenzhydrylamine resin, and hydrogen bromide as the cleavage and deprotecting agent for separating the resin-bound peptide from the resin support. In a particular aspect, the invention relates to the solid phase synthesis of thymosin .alpha..sub.1. The invention also relates to an improved process for synthesizing peptides, and particularly, thymosin .alpha..sub.1 and the thymosin .alpha..sub.1 -N.sub.1-14 fragment, by solid phase synthesis using an improved cleavage/deprotecting composition, namely hydrogen bromide with a mixture of anisole and thioanisole.
2. Description of the Prior Art
The function of peptides in human health has received much recognition in recent years, see e.g. "Peptides: A Medical Rediscovery" by Joseph Alper, High Technology, Vol. 3, No. 9, September 1983, pages 60-63. Peptides have been shown to function, for example, as hormones for regulating growth, reproduction, and immunology, and as neurotransmitters. While actual and positive biological results have been observed for several dozens of different peptides, still much work remains to be done to determine how the petides work in the body. For this purpose, large quantities of the peptides are required. Also, in order for the peptides of known biological activity to be of practical significance in health care, they must be made readily available in large quantities and at relatively low cost.
In addition to the "natural" peptides, i.e. those produced in the body, "synthetic" peptides or analogs, which may be considered as derivatives of natural peptides, but having one or more chemical modifications in the molecular structure of the natural peptide, are also valuable for their modified chemical properties, such as resistance to enzyme degradation. Here again, the availability of relatively inexpensive means to rapidly and inexpensively produce synthetic peptides would be highly valuable.
Presently, various procedures for synthesizing peptides, i.e. for formation of a peptide linkage between amino acids, are known. Conventional procedures include both solution-phase (or liquid phase) methods and solid-phase methods. A general discussion of peptide synthesis can be found, for example, in Schroder E. Lubke: "The Peptides," Vol. 1 (1966), Academic Press, New York, U.S.A, and Neurath and Hill, "The Proteins," Vol. 2 (1976), Academic Press, New York, U.S.A. Generally, these procedures involve the reaction between the free amino group of an amino acid or residue thereof having its carboxyl group, hydroxyl group or other reactive group(s) protected, and the free primary carboxylic group of another amino acid or residue thereof having its amino group or other reactive group(s) protected to form a peptide bond. Each amino acid in the desired sequence can be added singly successively to another amino acid or residue thereof or separate peptide fragments with the desired amino acid sequence can be synthesized and then condensed to provide the desired peptide.
In the past ten to twenty years the synthesis of peptides has been simplified by utilizing the solid phase synthesis method according to the general principles developed by Merrifield, [R. B. Merrifield, J. Am. Chem. Soc. 85, 2149-2154 (1963); Stewart, et al, Solid Phase Peptide Synthesis, Freeman & Co., San Francisco, CA (1969); Barany, et al, The Peptides, Analysis, Synthesis and Biology, Vol. 2, pages 1-284, (1980)]. Briefly in solid-phase peptide synthesis (SPPS), the carboxyl group of the first amino acid in a peptide is chemically bound to the surface of tiny, insoluble beads, with the other reactive sites on the amino acid being temporarily blocked. The amino acid-bound resin beads are placed in a reaction vessel and unbound acid is washed away. After chemically unblocking the reactive amino group, the next amino acid in the desired sequence of the object peptide is added with a chemical coupling agent such that the two acids bind together, via a peptide bond. The synthesis is continued by repeating the foregoing process with successive amino acids in the sequence being added one at a time until the total peptide sequence is built up on the resin. Upon completion of the desired peptide sequence, the protected peptide is cleaved from the resin support, and all protecting groups are removed. The cleavage reaction and removal of protecting groups may be accomplished simultaneously or sequentially.
SPPS has recently been adapted to automated and computerized instrumentation offering substantial improvements in speed, efficiency, and reliability. However, the use of such instrumentation has been substantially limited to production of peptides on a relatively small scale, on the order of a few milligrams per production cycle. Also, the procedure inherently results in the production of unwanted by-products, unreacted acids, solvents, coupling or decoupling agents, cleavage products and so on, making the subsequent purification procedures troublesome.
Although the art is aware of various different materials used as the insoluble beads, including glass, silica, synthetic resins, etc., the synthetic resins are most commonly used as the support material (insoluble beads). Conventional materials used as the resin include the styrene-divinyl benzene resin modified with a reactive group, such as chloromethylated styrene-divinyl benzene resin and benzhydrylamine resin (BHA). BHA is particularly useful for synthesis of peptide amides, i.e. peptides of which the C-terminal amino acid has a carboxylamide (--CONH.sub.2) group, since the amide group can be formed directly. The benzhydrylaminopolystyrenedivinyl benzene resin support is described by P. Rivaille, et al, Helv. Chim. Acta., 54, 2772 (1971).
Hydrogen fluoride, HF, is commonly used as the cleavage agent, and also as the deprotecting agent for removing the various blocking groups.
Unfortunately, HF, whether in liquid, anhydrous, or gaseous form, is highly corrosive and toxic requiring special handling and special plastic materials. As a result, the scale-up of the SPPS procedure from the laboratory scale to commercial scale production (e.g. on the order of one or more grams per production run) has proven extremely difficult.
It has been known to use the less corrosive and less toxic hydrogen bromide (HBr) is place of HF as the cleavage agent, alone, or together with anisole as a catalyst for cleaving the bound protected peptide from the supporting resin. However, HBr alone or with anisole does not function effectively as a cleavage agent with BHA resin, apparently due to the relatively high acid stability of BHA resin.
Accordingly, there is a great need to provide a method whereby peptides can be produced economically and simply using chemical substances which are non-corrosive to ordinary laboratory glasswares and of relatively low toxicity and which can produce the peptides on a commercial scale.